Method for classifying biological cells

ABSTRACT

A means of probing a biological cell sample with a optical source to determine the characteristics of the cell image by way of measuring parameters from its two dimensional Fourier transform. These techniques lead to a method of measuring discriminating parameters for cell classification.

This is a continuation, of application Ser. No. 581,799 filed May 29,1975, now abandoned.

FIELD OF THE INVENTION

This invention relates to an apparatus and a method for automaticallydetecting cell irregularities such as may be caused by cancer.

SUMMARY OF THE INVENTION

A widely used technique for reliably analyzing smears of body fluids isdescribed in a publication entitled, Diagnosis of Uterine Cancer by theVaginal Smear, by G. N. Papanicolaou and H. F. Trout, published byCommonwealth Fund, New York, 1943. In brief, Cancer cells which areexfoliated into body fluids are detectable upon microscopic analysis ofstained smears of the body fluid by highly trained personnel. It haslong been known as the Papanicolaou technique. Such technique involveshighly trained personnel employed in a time consuming and tediousobservation under microscope of a cell's:

(1) Nuclear Diameter,

(2) Cytoplasmic Diameter

(3) Nuclear Shape,

(4) Nuclear Chromatin,

(5) Cytoplasmic Stain Density,

(6) Nuclear Inhomogeneity,

(7) The relative parameters of the cell with respect to others and

(8) Cell Isolation.

The procedure employed was designed by a group of experts in the fieldof medicine.

As may be readily appreciated, the benefits of preventive medicine,especially in the case of cancer where early detection in many casesdetermines life or death, has led many to attempt to automate the cellclassification process. One known prior art attempt is found in the U.S.Pat. No. 3,327,117 issued June 20, 1967 to a Louis A. Kamensky, which isdirected to a means to sense the nucleus and cytoplasm changes that takeplace in a cancer cell from that of a normal cell by the use of anapparatus to probe the cell with an ultraviolet light energy of two wavelengths of a pre-determined order such that cell classification can beprovided by electrical signals dependent upon the absorbtion profiles ofboth wave lengths by the cell under study.

Another prior art attempt of automating cell classification is shown byU.S. Pat. No. 3,497,690 issued Feb. 24, 1970 to Leon L. Wheeless Jr., etal. In this attempt the cells are stained by a Fluorochrome, rather thanby the Papanicolaou technique, and subjected to an ultraviolet lightsource with subsequent measurement of the fluorescent response of thecell structure to provide its classification.

A principal object of this invention is to bring to the art of researchin cytology and pathology the investigatorial attributes of Fouriertransformation, i.e. the use of the diffracted light from a cell imagethat may be collected optically in a single plane and electricallyprobed to obtain the spatial intensity in relation to its spectralfrequency.

As will be readily appreciated by those skilled in the art of optics theillumination of an object of a light source will diffract the light intoan optical pattern which has varying intensity at different points inthe pattern. Such a diffraction pattern has no direct resemblence to theobject, as an image, but is a collection of a series of overlappingdiffraction patterns, each pattern due to individual features of theobject. Such diffraction patterns are the result of the modification ordiffraction of the light by the various points or details of the objector cell image.

While it is conceivable to utilize measuring apparatus to compute andindicate the spatial frequency of such deflected light it isconsiderably less cumbersome to collect such diffraction pattern by alens or sensors and transform its various paths of light energy into asingle plane. Such is the function of a Fourier transform apparatus inthe art of optics.

This invention was conceived during an investigation that was studyingcoherent optical signal processing techniques for cytologicalinvestigations of, for example, exfoliated cells. These investigationsfocused directly on the primary morphological features of the cell.

The exfoliated cells are found in body fluids such as secretions fromthe female genital track, gastric fluid, sputum, or in various bodycavities such as pleural fluid, peritioneal fluid, urine, cerebrospinalfluids, punctates expirmates or washings from epitheial surfaces, aswere heretoforth collected from certain body sites for microscopicexamination. In addition, spontaneously exfoliated cells can besupplemented by cells obtained directly from certain organs by the useof suitable instruments. Such cells maybe employed for detection anddiagnosis of various pathological conditions.

Actually, as stated before, the invention is concerned with the problemof automating cytological evaluation by measuring heretofore subtledistinctions between test cell samples that fall within the capabilitiesof machine recognition.

More particularly, this invention relates to a process whereby cells, orhigh resolution cell images, as by photographs or other means, aresubjected to a coherent light source. The light is scattered, diffractedand/or refracted into a two dimensional Fourier spectrum of the cell orcell image. This will provide a variety of transform parametersfunctionally related to the cell diameter, nuclear diameter, nucleardensity and other cell features, such as clumping of nucleardeoxyribonucleic acid (D.N.A.) that in combination greatly enhance thediscriminatory capabilities of cell evaluation.

This invention, therefore, advances the prior art by the disclosure of ameans to use the diffraction and refraction properties of coherent lightfor cytological screening and sample enrichment.

Another way of stating the object of this invention is to provide anapparatus and a process that will afford discrimination orclassification of cytological samples by the computation and evaluationof various statistical discriminating functions that can be related tocell diameter, nuclear diameter, and nuclear density-all cytologicaldiscriminating parameters, as for example in tests for malignancy inexfoliated cervical cells as compared to normal cells, in cytopathology.

A still further object of this invention is to provide an apparatus anda process for the screening of cytological samples that will accommodateirregularities in cell shapes in studies of the cells.

Still another object of this invention is to provide apparatus and aprocess for screening of cytological samples whereby various statisticaldiscrimination functions may be calculated, displayed and recorded.

The readers attention is directed to the article, "The Use of CoherentOptical Processing Techniques for Automatic Screening of CervicalCytological Samples," by R. E. Kopp, et al. The Journal ofHistochemistry and Cytochemistry, Volume 22, No. 7, 1974, pp 598-604wherein a theory and some results of this invention has been set forthwithout discussion of the apparatus and processes to fulfill the aboveobjects and others that will appear from the following drawings anddetailed description of this invention.

DRAWING DESCRIPTION

With reference to the drawings accompanying this disclosure there isshown by:

FIG. 1, An isometric schematic illustration of a combination ofapparatus that was used in carrying out the invention hereof;

FIG. 1A, An isometric illustration of a specific form of a combinationof means that has used this invention showing the projection of acollimated light source to obtain a diffraction pattern for a cell to beclassified by a radial scan.

FIG. 1B, An isometric illustration of an apparatus used in thisinvention in companion with the apparatus of FIG. 1A in an angular scanfor measuring selective additional parameters to complete cellclassification;

FIG. 2, A frontal view of a solid state electro-optical detector, as maybe utilized in the apparatus and process of this invention,

FIG. 2A, A frontal view of a fiber optic detector as may be utilized inthe apparatus and process of this invention,

FIG. 3, An isometric view of yet another form of a combination ofapparatus, as may be created in a laboratory for carrying out theprocedures of this invention;

FIG. 3A, A cross sectional view of an optical transducer used with theapparatus of FIG. 3;

FIG. 4, A schematic block illustration of the apparatus of thisinvention;

FIG. 5, A schematic block form of apparatus to provide computedindication of cell classification parameters;

FIG. 6, and FIG. 7 Graphical illustrations of measured signaturefunctions from the apparatus of FIG. 1A for three normal and threemalignant cell structures measured by apparatus according to thisinvention;

FIG. 8, and FIG. 9, Graphical illustrations of yet another measuredsignature function from the apparatus of FIG. 1B, for a normal and amalignant cell structure according to this invention; and

FIG. 10, A graphical illustration of average measured parameters for twocell structures computed by apparatus according to this invention.

DETAILED DESCRIPTION

This invention is useful in the classification of cells, and it isparticularly described with respect to the diagnosis of the cancercells, whose various structure or morphological features, (sizes, ratioof nuclear diameter to cytoplasmic diameter, nuclear density, nuclearirregulatity) differ from that of a normal, non-cancerous, cellstructure.

As will be readily appreciated in the performance of this invention,cell tissue smears are obtained and stained according to thePapanicolaou technique then fixed on a glass slide. Thereafter, and withparticular reference to the embodiment illustrated in FIG. 1, aphotograph is made of the cell structure, as by the use of a Nikon Fcamera body mounted onto a Nikon Su Ke microscope equipped with atrinocular head. It has been found that photographs bearing a desiredcontrast can be obtained by use of a 40× objective lens and 10× ocularlens employed with a 1/2× relay lens to yield a 200× magnification atthe film plane, and by the recording of the cell measured on K649Femulsion, 35 mm film. The high resolution capability, large contrastratio, and large saturation density were found to be useful filmcharacteristics for this purpose. The film records were then developedwith D-19 developer for 10 minutes at 68 degrees Fehrenheit to achievethe large contrast ratio desired.

The developed film was then mounted within a film transport 10 having anopening 12 for the exposure of the film 14 to a coherent beam from thelaser 16 expanded to cover the film with a lens 17. The coherent lightis scattered by the cell image forming a two dimensional angular FourierTransform spectrum of the cell image 15. The angular spectrum is thenprojected by a lens 18 to a transform plane 28.

The Fourier transform or diffraction pattern from the lens 18 is thencollected by a magnifying lens 20 and projected on a face of an opticaldetector 22 that is adapted to provide electrical signals for contacts24 that will be indicative of various measured parameters of thediffraction pattern on the detector 22, as will be describedhereinafter.

This is also shown with reference to FIGS. 1A and 1B which shows analternative means of measuring various details of the diffractionpattern where the laser beam 26 generates the Fourier transform to adiffraction plane 28 that is expanded by the lens 20, as shown by theenergy lines 30 and 32 to project a diffraction pattern 34 on a mask 36.The mask 36 and photomultiplier 42 has been substituted in thisembodiment for the optical detector 22 of FIG. 1. As seen, mask 36 hasan aperture 38 that permits the passage of light energy of a portion ofthe diffraction pattern to the sensor lens opening 40 of aphotomultiplier 42, as will be readily familiar to those skilled in theart of devices of measuring light intensity. The photomultiplier 42 isprovided with electrical terminals 44 that are the equivalent ofterminal 24 for the feeding of the electrical signals generated thereby,whose amplitude is a function of the intensity of the light energytransmitted by the mask.

Another means of measuring other specific features of the diffractionpattern is shown in FIG. 1B. Here one takes angular scans of the lightintensity in the diffraction plane using a rotating mask 130 containinga small hole 132 located at a radial distance from the center of thediffraction plane. The mask was rotated mechanically by drive 136 ofmotor 134 through 360° and the light energy passing through the smallhole was collected by a means such as a collector lens 131 andphotomultiplier 42.

One may also combine the feature measuring capability of the twomechanically activated systems shown in FIG. 1A and 1B, by a singlesolid state electro-optical detector as shown in FIG. 2.

Specifically, there is shown in FIG. 2 a solid-state detector having anannular plate 208 comprising a plurality of semiconductor fabricatedareas layed out in geometric patterns. More particularly, the plate faceis divided into two semi-circular halves 210 and 212. There isfabricated by the use of the well known semi-conductor technology aplurality of individual rings, rings 214, 216, 218 and 220 beingspecifically called out by way of example, in the upper half 210. On theface of the lower half 212 there is similarly provided a plurality ofpartial wedge shaped active areas, wedges 222, 224, 226 and 228 beingcalled out by way of example leaving non-active surfaces 230 and 232 inthe lower semi-circular half 212. Conductors (not shown) lead from eachof the rings 214 etc. and wedges 222, etc. so as to provide for theconducting of an electrical signal in accordance with the light energyon these rings or wedges, as will also be readily familiar to thoseskilled in the art of light sensitive semi-conductor technology. Thedetector shown is an improvement upon the type as disclosed by U.S. Pat.No. 3,689,772. The improvement being the elimination of unwantedconfusion which would be caused by large area wedges.

It has been found that by this design of the detector 22 it is possibleto automate the process of cell classification to a greater degree thanis possible by the use of the processes involved with apparatus of FIGS.1A and 1B. This results from the fact that the rings 214, etc. beingeach able to provide an electrical signal indicative of the total energyabout the individual ring will provide a radial scan similar to thatpermitted by the mask 36; and the limited areas of the angular positionof the partial wedges 222, etc. will provide an electrical signalindicative of an angular scan of any light intensity similar to thatwhich will be provided by the use of the mask 130.

A fiber-optic detector may also be used in place of a photomultiplier ora solid-state detector. Such a fiber-optic detector is shown by FIG. 2Ato comprise a disc 229 mounting bundles of individual light conductingfibers 231, 233, etc. Each bundle of fibers, as will be readilyunderstood to those skilled in the art, will be connected withindividual photo sensitive means such as phototransistors (not shown) tofurnish individual electrical signals to a computer or similar measuringmeans to indicate in the same fashion as the other detector means thelight energy within the Fourier Transform pattern. Such a bundle ofoptical fibers can be connected in the measuring circuit in almost anydesired way to provide analysis of all or any part of the pattern byrendering all or some bundles effective in providing electrical analysissignals.

With reference now to FIGS. 3 and 3A there is shown a still further formof apparatus that embodies the principles of this invention. Moreparticularly, there is shown a stable platform 46 on which is mounted alaser 48, an expanding lens 49, a Fourier transform lens 50 and asolid-state electro-optical detector within a housing 52 that isconnected by leads 54 to a minicomputer 58 for taking the measurementsof the detector 52 and providing a computed indication thereof. In thisembodiment a projection microscope 60 is utilized to view the celltissues prepared, as aforesaid, and placed on the microscope stage bythe glass slide 62. As schematically shown, the projection microscope 60has a means 64 to focus light, such as a variable intensity projectionbulb, upon the glass slide 62 in accordance with a variable control 63,whereby a projection, as by a lens 70, of the cell image may bedelivered to a optical transducer means within a housing 72. At the sametime an operator can also view the glass slide material by means ofeyepiece lenses 118. The contrast of the image on the transducer may bevaried by variable resistance control 66 as shown by indicator 68. Theoptical transducer is more particularly described with reference to FIG.3A to consist of a sandwich between conductive coated glass flims 74 and76 of photoconductive layers 78 and 82 surrounding a layer 80 ofelectrically activated material with variable optical transmissionproperties. A power source 84, illustrated as a battery in FIG. 3A andprovided by means of an electrical outlet in FIG. 3 is connected byappropriate conductors to the transducer shown, as will be readilyappreciated by those skilled in the art. A more particular descriptionof a typical transducer such as may be required herein is shown by U.S.Pat. No. 3,732,429 issued May 8, 1973. In brief a photoconductivecadmium sulfide layer 78 functions to convert the incoherent light image90 passing through a beam splitter 88 also projecting the coherent lightenergy 86 of the laser beam 48 (see FIG. 3) projected on the conductivecoated glass film 74 into a flow of current through a liquid crystallayer 80. The liquid crystal 80 then responds to the current flow bychanging its optical transmission; in particular, it becomes translucentsuch that the image in the liquid crystal layer is impressed onto thelaser beam 86 after the beam passes through the device to provide anoutput beam 92 projecting a coherent image of the cell on slide 62through the Fourier transform lens 50. Therefore, one may view thetransducer as an instantaneously developed film transparency in that theprojection therefrom is the same as if one exposed a piece of film to anincoherent light image, placed the developed film transparency in a pathof a laser beam, and observed the outcoming laser beam diffractionpattern, as by the apparatus illustrated in FIG. 1 and 1A. Furthermore,the image transparency (density & contrast) may be varied by adjustingthe exciting voltage applied to it as by control 66.

A schematic illustration of the general means for generating and thenmeasuring cell diffraction patterns is shown in FIG. 4 and includes thevarious previously described system. The laser beam 94 from a laser 96,such as a Spectra Physics model 124A, is expanded with a lens and apinhole combination such as a 4.5 mm lens and 6.8 micron pinhole in aspatial filter 98, such as a Spectra Physics model 330. A mechanicalshutter 100 is employed between the filter 98 and a collimation lens102, such as a Tropel f-4, 200 mm lens, for exposure control. Aftercollimation, the center portion of the expanded beam is transmittedthrough a rectangular aperture (typically 25/32 in.×1/2 in.) formed by alight baffle 104, such as a Conductron four-way adjustable aperturemodel, to a film holder or optical transducer 106. If a film holder isused, it has been found that as individual frame of a 35 mm cell imagefilm strip can be held by clamping it between two optical flats, wettedwith a refractive index matching liquid. Thereafter, the lightdiffracted by the cell image is collected with a transform lens 108 toprovide a transform pattern 110. However, as the transform in the backfocal plane of lens 108 may be too small for convenient observation, ashort focal length magnifier 112 is used to enlarge the diffractionpattern image. In one embodiment of the optical system the magnificationfactor used thus far was about 40×, and the equivalent spatial frequencyscale in the cell image plane was about 1000 cycles/mm per cm. Themagnified diffraction pattern 114 (34 in FIG. 1A) is then projected uponappropriate detector system 116 as described above.

With the aforedescribed measuring equipment there is permitted therecording of signals from radial and angular information of the Fouriertransform spectrum upon a magnetic tape for data storage and handling.Reference should now be made to FIG. 5 showing that subsequent to therecording on the magnetic tape such tapes may be digitized by aninstrument 190.

Final processing of the raw, digitized data is achievable by a computer204 and at the same time provided to a magnetic tape machine 206 topermit retention of the digitized data for subsequent checking of thecomputation, if necessary.

As will appear hereinafter, the aforesaid apparatus will permit thegeneration of the graphical illustrations of FIGS. 6 through 10 frommeasured data for the comparison of a normal cell with a malignant cell.At the same time the apparatus provides signals that may be utilized ina statistical analysis that will utilize the fundamentals of variousstatistical decision procedures such as the Baysian decision process inpattern classification.

Now in particular regard to the process that is only permitted by theaforedescribed apparatus in the classification of cell tissue, and moreparticularly exfolitated samples such as are presently subjected tomicroscopic examination in the screening of cervical cell samples fordetermination of cancer, the normal process hereof involves first thestep of the optical generation of a two dimensional Fourier transform.This involves the use of the cell image 15 (see FIG. 1) to spatiallymodulate the collimated laser beam thereby causing a diffraction ofcoherent light which is then collected by a Fourier transform lenslocated one focal length behind the image plane.

At a distance of one focal length behind the lens, the transversespatial distribution of optical energy is functionally related to themodulated light immediately behind the film. This relationship is thetwo-dimensional Fourier transform given by:

    F(w.sub.y,w.sub.y)=∫∫f(x.sub.o,y.sub.o) e .sup.-j (w.sbsp.x.sup.x.sbsp.o.sup.+w.sbsp.y.sup.y.sbsp.o.sup.) dx.sub.o dy.sub.o (1)

where transform size scaling is determined by the wavelength (λ) of thecoherent light and the focal light (f) of the lens:

    w.sub.x =2πx.sub.f /λf, w.sub.y =2πy.sub.f /λf (2)

where F is the Fourier transform of the cell image f, and the variablesx_(f) and y_(f) are the cartesian coordinates in the transform plane.

There are several properties of the Fourier transform relationship whichprompt the use of this approach as a screening device. The transversespatial distribution, _(F) (w_(x), w_(y)), is centered on the opticalaxis of the lens, with 180° symmetry in the transform plane, and itsamplitude is independent of the location of the cell image on the film.This circumvents the problem of searching for a particular image orfeature of the image within the field of view of an instrument. Similarsized features in the cell contribute energy over the same region of thespectrum with small sensitivity to their precise shape and independentof their location within the image. Although it is not readily apparentfrom Eq. (1), there is an inverse relationship between the size of theimage and the region of the Fourier spectrum containing the energydiffracted by the image--that is, small objects have large spectra andlarge objects have small spectra. These properties are attractive froman instrumentation point of view when considering a screening device. Itis again worth mentioning that the relationship given in Eq. (1) is nothighly sensitive to cell image motion along the optical axis, as in thecase in scanning systems which require elaborate automatic focusingsystems.

A more complete discussion and analysis of the optical generation of twodimensional Fourier transform can be found by reference to the booksINTRODUCTION TO FOURIER OBJECTS by J. W. Goodman, McGraw Hill, N.Y.1968, pp. 77-83 and AN INTRODUCTION TO COHERENT OPTICS AND HOLOGRAPHY byG. W. Stroke, Academic Press, N.Y. 1966, pp. 70-96.

The Fourier transform pattern of all the cells classified to date weresubsequently analyzed quantitatively by measuring both the averageradial distribution of light enery in the transform and also the angularvariation at selected radial positions in the transform plane. Theaverage radial energy distribution was measured using the apparatusshown schematically in FIGS. 1A and 1B. A spectrum sampling mask wasmade consisting of an open annulas 38 of radius r_(o) and width Δr. Thismask was located after the Fourier transform lens 18 and in front of aphotomultiplier tube 42 which measured the integrated light intensitywithin the annulus. The mask and photomultiplier were then moved inunison along the optical axis of the transform lens, i.e., along thex-axis in FIG. 1A, and the outputs recorded as a function of x. As theorigin of the x-axis is located at the point where the rays of lightconstituting the transform have an apparent focus, the spreading bundleof these rays has a distribution of intensity along any plane (definedby x-x_(o)) that is given approximately as ##EQU1## where F (ρ,θ) is theFourier transform expressed in polar coordinates ρ,θ and ρ² =w_(y) ²+w_(y) ² ; r the radial position in the spreading bundle of rays; andthe factor (ar/xo) contains the scale factor "a" of the optical systemindicating the linear dilation of the beam, and the factor 1/(x_(o) ²+r²) accounts for the usual squarelaw fall-off of the light intensity.The output of the photomultiplier M_(out) is proportional to theintegrated light coming through the annulus in the mask when it islocated at an arbitrary position, or explicitly: ##EQU2## It may benecessary to have webs to support the annulus in the mask. The webs willblock some of the light. (However, the webs are useful in obscuringlight in the diffraction pattern due to the rectangular aperturegeometry).

To gain some insight into the nature of this measurement we will assumethat the annulus width Δr is sufficiently small so that: ##EQU3## wherethe average pertains to the average taken over the angular variations.If r_(o) and Δr are fixed and x is varied we may sample the average |F|²at different values of radial spatial frequency, ρ. Thus assuming thatthe optical scale factor "a" is selected so that:

    (ar.sub.o /x)=ρ                                        (6)

we find that: ##EQU4## Finally, with a spatial frequency satisfying ρ<<awe have: ##EQU5## or an output that a proportional to ρ² times theintensity squared of the transform.

Different masks were constructed having Δr/r_(o) ratios of 0.25 and 0.1and were used to measure ρ² |F|_(avg) ² over two spatial frequencyranges: 10 to 100 cycles/mm; and 100 to 1000 cycles/mm, respectivelyreferred back to the microscope slide. The data was reduced further andnormalized by dividing each output by the total light energy E in theSpectrum to complete the step of conducting a radial scan of thediffraction pattern. In such scan runs data from one of the masksproduced the curves of FIGS. 6 and 7. These curves are measures of theaverage radial distribution of normalized energy in the transforms andone may regard them as measures of ρ² |F|_(avg) ², bearing in mind theabove, particularly the fact that the width of the annulus is notarbitrarily small but in fact Δr/r_(o) ≈0.25 or 0.1 with the greatesterror appearing in the low frequencies. Such curves may be regarded as asignature functions for the particular cell.

In addition to the annulus data, angular scans of the light intensity inthe Fourier transform were made with a masking containing a small hole132 located at a radial distance from the center of the transform.Actually this step includes placing the hole at four different radialdistances corresponding to spatial frequencies of 430, 485, 565 and 630cycles/mm. The hole diameter corresponds to 65 cycles per millimeter.The mask was rotated through 360°, measuring the optical energy passingthrough the small hole as a function of the angular position in theFourier transform. This was done for each of the four radial distancesmentioned. The curves of the angular variations are also shown for thecells by FIGS. 8 and 9 and may be regarded as another signature functionfor the cell. The ordinate in each case is in arbitrary relative scale,nevertheless the results can be compared from curve to curve since themaximum value on each scale is related to the others in the set.

It should be noted that the curves of FIG. 6 are the result of the useof the apparatus of this invention in screening normal cell tissue;whereas the curves of FIG. 7 are a similar result in the screening ofmalignant cell tissue. The concave shape of the curves of FIG. 6 for thenormal cells is readily contrasted with the convex shape of the curvefor the malignant cells, thereby providing a means to distinguish normaland malignant cells by the slope of their radial distribution curve in anegative slopes and malignant cells having positive slopes.

With more particular regard to FIGS. 8 and 9, angular scans of malignantcells (shown by FIG. 9) tend to have larger variations than the scansfor the normal cells as shown by FIG. 8. This was investigated furtherby means of a power spectral analysis of the angular scan curves. Withreference to FIG. 10 there is shown the average power spectrum for agroup of malignant cells (trace 236) and a group of normal cells (trace238). It should be noted from FIG. 10 that there is a range illustratedbetween the dotted lines 240 and 242 where one can find a wideseparation of the average power spectrum density for malignant andnormal cells within which machine measureable features would be lesssubject to classification errors than in other areas of the averagepower spectrum density for these cells.

Data collected from a group of cells was examined extensively andvarious parameters were abstracted from this data to be used fordiscriminating cell features. These parameters or features included thetotal light energy in the transform of the cell, the slope of ρ²|F|_(avg) ² (the slope of the product of the square of the radialfrequency times the average radial distribution of light energy in thetransform), the variance of ρ² |F|_(avg) ² over a range of valves of ρ,the variance of |F|² over angular coordinates, and the total energy in aportion of the power spectrum of the angular variation of |F|², etc. Tobe explicit, some of the parameters are given by the expressions:

Total energy: ##EQU6## where (ρ represents the radial spatial frequencycoordinate, θ the angle, and F(ρ,Γ) the Fourier transform distributionin polar coordinates):

Slope of ρ² |F|_(avg) ² in the range a thru d (cycles/mm) normalized tototal energy: ##EQU7## where a, b, c and d are specific radial positionsof the selected range of radial spatial frequency, such as 10, 20, 30and 40 cycles/mm.

Variance of ρ² |F|_(avg) ² in a second range e to f cycles/mm normalizedto total energy: ##EQU8## where e to f is the specific radial positionof the beginning and end of the second range selected such as 10 to 100cycles/mm.

Variance of angular distribution of intensity in range g to h cycles/mm:##EQU9## where g to h is the specific radial position of the beginningand end of the third range selected such as 500 to 600 cycles/mm.

Total energy in a portion of the power spectrum of the angulardistribution of intensity in the range from g to h cycles/mm: ##EQU10##where i to m are adjustable portions of the power spectrum such as overthe 12^(th) to 19^(th) harmonic, and ##EQU11##

In the case where the solid state detector of FIG. 2 was employed tomeasure the features, we must approximate the various parameters definedabove using the sampled ring and wedge outputs from the detector. Thusthe ring outputs of the detector are: ##EQU12## where ρ₁ (I) and ρ₂ (I)are the inner and outer radii of the I^(th) ring, and the wedge outputsare: ##EQU13## where θ₁ (I) and θ₂ (I) are the angular coordinates ofthe sector forming the wedge and 600 and 500 are two frequency ranges.The fact that the Fourier plane detector presently in use provided a"sampled output" of the transform of the cell images required a varietyof approximations to be made to obtain approximate values for the fivedesired features. For example, to approximate the desired integrals ofρ² |F|² used in the calculations of slope, we approximated: ##EQU14##where for the particular detector in question ring's 5 & 6 spanned therange of ρ from 30 to 40 cycles/mm which implies that the approximation:##EQU15## was used. Similarly in the variance of ρ² |F|², we used theapproximation ##EQU16## which assumed ##EQU17## In a similar way we used##EQU18## to define the angular distribution of intensity to be used inobtaining the last two features. Finally, in lieu of measuring the totalenergy in the specified portion of the spectrum of the angular data bycomputing the Fourier coefficients, a_(n), a digital filtering techniquewas employed. Specifically, a digital filter operation was formed thatused a four-pole Butterworth approximation to a band-pass filter withpass band from the 12^(th) through 19^(th) harmonic. The angulardistribution or wedge data was then filtered, and the variance of thefiltered signal was obtained. (The variance was used rather than thetotal energy of the signal to improve the approximation--with an idealband-pass filter, the output should have zero mean.)

Then, as will be obvious to those skilled in the art, the following sixfeatures were computed from the detector output where five features areapproximations to the basic five features identified previously, and asixth (the third listed below) was an attempt to use a simplifiedversion of the variance of the radial information: (the expressions usedapplied to a specific solid state detector manufactured by RecognitionSystem Incorporated which had 32 rings and 32 wedges. Moreover a mask asshown in FIG. 2 was placed over the detector so that only the portion oflight in the range of 500-600 cycles/mm fell on the 32 wedges).

(1) Total energy: ##EQU19##

(2) Slope of ρ² |F|² in the range 10-40 cycles/mm normalized to totalenergy: ##EQU20##

(3) Variance of radial intensity from 10-100 cycles/mm normalized tototal energy: ##EQU21##

(4) Variance of ρ² |F|² in the range 10-100 cycles/mm normalized tototal energy: ##EQU22##

(5) Variance of angular distribution of intensity in the range 500-600cycles/mm: ##EQU23##

(6) Total energy in a portion of the spectrum of the angulardistribution of intensity in the range from 500-600 cycles/mm: ##EQU24##where ##EQU25## These expressions are programmed into a computer such ascomputer 58 (shown in FIG. 3).

Finally in carrying out this invention it is desired to set forth themethods of analyses used to study the data collected, particularly thestatistical techniques used to investigate the discriminating capabilityof the various features. The statistical techniques include the Baysiandecision theory for multivariate Gaussian process, and the concept of"divergence" as a measure of the performance of a specific feature orgroup of features. These techniques are described in full in suchreferences as INFORMATION THEORY AND STATISTICS, by Kullback, S., JohnWiley and Sons, London 1959, and the article by Marill, T., Green, D.M., "On the Effectiveness of Receptors in Recognition Systems," IEEETransactions On Information Theory, pp. 11-17, January 1963. Forexample, there was employed a quadratic discriminant function thatresults from a Baysian decision rule to form the basis of a decisionrule. Thus if P1 and M1 are the covariance matrix and mean vector forthe class of normal cells whereas P2 and M2 are the correspondingvariables for the class of abnormal cells, then we may establish adecision function defined by

    Q(X)=(X-M1).sup.T P1.sup.-1 (X-M1)-(X-M2).sup.T P2.sup.-1 (X-M2)=C

which would allow one to compute a number C for each cell depending uponthe feature vector X measured for that cell. If the value of C for acell is above some pre-defined value (decision level) than one would saythat the cell is abnormal, and if it is below the level it would causeone to call the cell normal. In the problem of cell and smeardiscrimination, the value of the decision level can be adjusted toparametrically determine the statistical occurrence of errors ofmisclassification, i.e., the relative values of the false positive(normal cells misclassified as abnormal) and false negative (abnormalcells misclassified as normal). Other statistical decision proceduresboth linear and nonlinear may also be utilized such as the FischerDiscriminant described in the previous reference.

A computer program was written to carry out the Baysian discrimination,as well as compute the divergence number (a measure of power of featuresto discriminate cells) for the features being used. The specificcomponents to be used for the pattern vector are requested and thestatistics are calculated in the same manner as in the divergenceanalysis program. The means, standard deviation, scaling and correlationcoefficients can be printed. The values of the Baysian quadraticdiscriminating function, as well as the Fischer Discriminant were thencalculated and printed along with the maximum and minimum value for eachclass. A listing of the program and a typical printout per the method ofthis invention for the cells of FIGS. 7 and 8 was, as follows:

    ______________________________________                                        Divergence = 66.0428                                                          Difference of weighted distances from mean vectors                            Cell No.  Quad. Dist.   Normal      F. Dist.                                  ______________________________________                                        177       -22.8333      ↑     -5.1745                                   148       - 7.2503      ↑     -3.2750                                   187       - 4.8001      ↑     -1.7051                                   Cell No.  Quad. Dist.   Malig.      F. Dist.                                  ______________________________________                                        173       59.6674       ↓    3.2854                                    174       30.2046       ↓    2.2887                                    175       27.5866       ↓    0.0744                                    Divergence with Lin. Disc.  15.5274                                           ______________________________________                                    

OPERATION

Therefore, with the aforedescribed apparatus and theory this inventionenabled the selection of various cell diffraction pattern features suchas slope, energy and angular variance individually or collectively.

More particularly, a source of collimated light energy, as from a laser16 or other source of coherent light is used to illuminate a cell imagein a film holder or a real time transducer to generate an intensityprofile of the cell structure. Actually, the image of the cell spatiallymodulates the collimated laser beam causing diffraction of the coherentlight.

The next step of the process of this invention utilizes the Fouriertransform lens 18 to collect the diffracted coherent light at a locationbehind the lens to create a diffraction pattern whose transverse spatialdistribution of optical energy is functionally related to the modulatedlight immediately behind the image as expressed by equation (1). Thelocation of the lens was established to be a distance of one focallength behind the image which then resulted in the establishment of theFourier transform or diffraction pattern at a distance of one focallength behind the lens 18.

Since the diffraction pattern in the back focal plane, i.e. transformplane, may be too small for convenient observation, a short focal lengthmagnifier 20 is then used to enlarge the diffraction pattern and projectit in an enlarged area.

From this point the process can continue by one or the other of severalknown ways to classify the cell structure's diffraction pattern basedupon its optical energy. In one such process the apparatus of FIG. 1 isemployed to collect the diffraction pattern on the face of a solid-stateelectro-optical detector 22. With this apparatus the process ofclassification involves the creation of electrical energy to outputterminals such as the two terminals 24 shown. A comparison of theelectrical energy from each of the rings and from each of the wedgeswill provide not only signals of the optical energy but of the radialand angular location of same as will be further explained in referenceto the process to be employed with the apparatus of FIGS. 1B and 4.

However, before entertaining an explanation of that apparatus it is alsopossible with the apparatus of FIG. 4 to obtain a photograph of theoptical energy of the diffraction pattern for cell classification. Moreparticularly, the diffraction pattern its collected upon a detector 116that may simply be a camera back screen. In the case of screeningexfoliated cervical cells to determine if they are cancer cells withwhich the apparatus and processes of this invention has been usefullyemployed it is possible to see distinctions in such spectral photographsbetween normal and cancer cells. Specifically, the spectral photographof a normal cell will have angular symmetry of a "bulls-eye" pattern.characteristics of a transform of a circular opaque object. This readilycontrasts with the broken or mottled pattern of the transform of aninvasive cancer cell. In that the rules of the Patent Office do notpermit the use of photographs in drawings of a patent, reference is madeto the article in "The Journal of Histochemistry and Cytochemistry"aforementioned for a picture of what is possible by this apparatus. Toclassify the cell structure by this method only requires the viewing ofthe spectral photograph. It is also within the state of the art to use areal-time (optical) transducer, such as shown by FIG. 2A, to view theFourier transform of the cell immediately, as it is projected frommagnifier 112. It is also possible under the state of the art togenerate the Fourier Transform pattern directly from the cell itself.

Finally with reference to the operation of the apparatus of FIGS. 1A and1B in obtaining a process of cell classification according to thisinvention the only apparatus and processes therefrom which differ fromthat aforedescribed is in reference to the use of masks 36 and 130 incombination with a photomultiplier 42. With this apparatus diffractionpattern 34 is projected by the enlarging microscope 20 on the face ofmask 36 or 130. Mask 36 transmits by way of aperture 38 the spatialfrequency spectrum of this diffraction pattern 34 to the ground glassscreen of the photomultiplier 42. The mask 36 being mounted on frame 136that is translatable along the X (optical) axis will transmit the radialvariations of the spatial frequency spectrum which is one that expandsin a cone fashion as the energy lines 30-32 indicate. As thephotomultiplier 42 is moved in unison with the frame 136 for mask 36along the optic axis, a point in the annulus in effect scans thediffraction pattern radially. During this multiposition scanning thepotentiometer 144 provides a measurement of the spatial frequency scalewhich with the signals of light intensity of photomultiplier 42 willpermit a graphical illustration of these scans as shown for the variouscells of FIGS. 6 and 7 in according to equation (8).

Thereafter the mask 130 is substituted for mask 36 and driven by a motor134 to revolve, as in a preferred form of apparatus, to provide angularscan data using a scanning hole at a predetermined frequency (as perexample) 630 cycles per millimeter. This, as previously stated willprovide a scan of optical energy passing through the hole as a functionof the angular position in the Fourier transform; and an output signalof same from the photomultiplier according to the expression of equation(8). This would permit graphical illustration of the optical energy asper FIGS. 8 and 9 or 10, above described, to be related to a normal celland an invasive cancer cell.

As may be appreciated by those skilled in the art the graphicalillustrations may be provided by automatically plotting theradial/angular frequency content versus the spatial frequency/harmoniccontent by a plotter (not shown) in conjunction with the computer 204providing discriminating readouts as aforesaid. It should also beappreciated that the apparatus for the process described herein may takestill other forms without departing from the scope of this invention asset forth by the appended claims:

We claim:
 1. A method for recognizing normal and cancerous cellstructures, said method comprising the steps of:obtaining a diffractionpattern of light representative of a cell image; generating firstsignals from the diffraction pattern of light of a level of radialenergy over a preselected limited frequency range from selected portionsof said diffracted pattern of light and from the total energy of saiddiffraction pattern of light; generating second signals of eachfrequency used to obtain signals of a level of radial energy at eachindividual frequency used; generating a sequence of signalsrepresentative of a multiple of first signals of level of radial energyand the square of said second signals of the individual frequency usedwhere said first signals are derived; generating a third signalrepresentative of the slope of the sequence of signals; and displayingnormal or cancerous indication from differences in said third signalbetween cancerous and normal cell structures of the slope of saidsequence of signals representing radial intensity at a radial frequency.2. A method of probing a Fourier transform of a cell image to observecertain cell discriminating features comprising:producing on an opticaltransducer a cell image; directing a coherent light source through thecell image in the transducer; projecting a diffraction pattern of thecell image in the form of light energy from the transducer to anelectrical signal generating device that uses light energy to generateelectrical signals; generating signals representative of a level ofradial light energy over a frequency of 10-40 cycles/mm, said generatingof signals being productive of a signal from light energy at each radialfrequency in the range aforesaid and the individual radial frequency atwhich obtained; generating a sequence of signals by multiplying thesignal of light energy at each radial frequency by the square of thesignal of the said individual frequency between 10-40 cycles/mm at whichobtained starting at 10 cycles/mm and ending at 40 cycles/mm; obtaininga signal representative of the slope of all the sequence of signals; andactivating an indicator by the signal representative of the slope inillustrating cell discriminating features so that one can readilyobserve differences thereof with a model cell.
 3. The method ofrecognizing normal and cancerous cell structures characterized as aprocess of delineating differences between normal and cancerous cellscomprising:illuminating a cell image with a source of collimated lightenergy to generate an intensity profile of the cell structure;generating a Fourier transform of the intensity profile of the cellstructure; generating a radial signature signal from the Fouriertransform as a first electrical signal representative of the product ofthe square of the radial spatial frequency times the average intensityof the transform, the radial signature signal being obtained from aportion of the transform characterized by,

    ρ.sup.2 |F(ρ,θ)|.sub.avg.sup.2

where F(ρ,θ)=the Fourier transform of the cell's image expressed inpolar coordinates ρ=radial spatial frequency θ=polar angleand theaverage is taken over the polar angle variable;generating an angularsignature signal from the Fourier transform as a second electricalsignal representative of the polar angle variation of the energy in aspecified radial band of a transform plane, the angular signature signalbeing obtained from a portion of the transform characterized by,##EQU26## where g and h are the radial spatial frequency dimension ofthe radial band; generating a total energy signature signal (E) from theFourier transform as a third electrical signal representative of thetotal energy in the Fourier transform, the signal (E) being obtainedfrom a portion of the transform characterized by, ##EQU27## andillustrating information with the aforesaid first, second and thirdelectrical signals whereupon recognition of cell structure is possible.4. The method of claim 3 and further characterized by;generating withsaid first electrical signal a signal of the slope of a normalizedradial signature signal by selecting first a range of radial spatialfrequency of the Fourier transform so as to obtain signals proportionalto the slope of the product of the square of the radial frequency timesthe average radial distribution of light energy in the transform in theselected range, said slope being located from the transform according tothe expression, ##EQU28## where E=total energy signature parameter inthe transform a,b,c,d=specific radial positions of selected range ofradial spatial frequency;generating also with said first electricalsignal a signal to measure the fluctuations of the normalized radialsignature signal by selecting a second range of radial spatial frequencyof the Fourier transform so as to obtain signals proportional to thevariance of the product of the square of the radial frequency times theaverage over the polar angle of the radial distribution of light energyin the transform across said range divided by the total energy signatureparameter, said variance being located from the transform according tothe expression, ##EQU29## where e and f=selected radial positions of thesecond range selected; generating with said second electrical signal asignal to measure the fluctuations of the angular signature signal byselecting a third range of radial spatial frequency and a Δθ so as toobtain signals proportional to the variance of angular distribution ofenergy in the Fourier transform in said third range, said variance ofangular distribution being located in the transform by the expression,##EQU30## where g and h=selected radial positions of the beginning andend of the third range selected and Δθ=an increment in the polarangle;generating with said third electrical signal signals proportionalto the total energy in a portion of the power spectrum of thedistribution of the Fourier transform in said third range of spatialfrequency, said total energy being from a portion of the transformlocated by the expression, ##EQU31## where i and m=adjustable portionsof the power spectrum over selected specific harmonics and ##EQU32##printing information using said signals to illustrate indicators ofdiscriminating values enabling recognition of normal and cancerouscells.
 5. A method for recognizing normal and cancerous cell structurescomprising:using a coherent light source to obtain a diffraction patternof light energy representative of a cell structure; focusing thediffraction pattern of light energy on a device to generate electricalsignals therefrom; generating signals representative of a level ofradial energy from the diffracting pattern light energy over a frequencyrange of 10 to 40 cycles/mm, said signals being generated so as toprovide separate signals representative of the level of radial energy ateach spatial frequency in said frequency range of 10 to 40 cycles/mm;generating a signal representative of the square of each spatialfrequency; generating a sequence of signals by multiplying each separatesignal of the level of radial energy by the signal representative of thesquare of the spatial frequency where said level is being taken startingwith the spatial frequency of 10 cycles/mm and ending with 40 cycles/mm;generating a signal of the slope of the sequence of signals; andactivating a display with the signal of slope to automatically indicatewhether a cell is cancerous or not.
 6. The method of claim 5 and furthercomprising the steps of generating signals representative of the radialenergy distribution from 10 cycles/mm to 100 cycles/mm, said signalsbeing generated so as to provide separate signals representative of thelevel of radial energy at each spatial frequency in said frequency rangeof 10 to 100 cycles/mm; generating a signal representative of thevariance of the product of the signal representing the square of thespatial frequency and the signal representative of the level of radialenergy at each said spatial frequency; generating a signal of the totalenergy of the diffraction pattern light energy; generating a signal ofthe dividend of the signal of the variance of the product and the signalof total energy; and applying the signal of the dividend to theapplication of the display for refined definition of display operation.